Many proteins of industrial and medical interest can effectively be produced by introducing the corresponding genes into microorganisms using genetic engineering techniques. A favorite microorganism for such production is Escherichia coli, whose genetic makeup is probably the best known of all organisms, and whose mutants lend themselves to elaborate manipulations.
Production of a protein in E. coli can yield very large amounts of protein, which must usually be purified to remove other proteins and bacterial components (including bacterial endotoxin).
It is desirable to devise a system such that the cells of E. coli will produce a desired protein and release it into the medium, thereby facilitating purification. With such a system, it may also be possible to collect secreted proteins in large amounts from E. coli cells growing under continuous flow conditions.
The kil gene is identified as an open frame in plasmid pColE1,the source of colicin El. The MIT group (Drs. S.E. Luria and J.L. Suit) have demonstrated the existence and the role of the kil gene: Expression of an intact kil gene located under the ColE1 promoter causes cell death and release of colicin El from the bacteria (Suit, et al. [1983]J. Bacteriol. 161:944-948). Colicin El, like all colicins, is produced in the cytoplasm, and is not located in the periplasm. Several other colicin-producing plasmids have been found to contain genes which function analogously to that of kil in pCo1E1 (Sabik, J.F., Suit, J.L. and Luria, S.E. [1983]J. Bacteriol. 153:1479-1485. The colicin-kil promoters in all these plasmids are inducible by treatment with mitomycin C. The protein product of kil has not yet been isolated; it is believed to be made as a peptide of 45 amino acids, which may be processed in vivo by proteolysis.
When the kil gene is expressed under a strong promoter, kil.sup.s cells of E. coli are rapidly killed. Recently Kobayashi et al. (Kobayashi, T. et al. [1986]J. Bacteriol. 166:728-732) reported that weak expression of kil (under an unknown weak promoter) causes release of two periplasmic proteins (.beta.-lactamase and alkaline phosphatase) with little cell killing.
Recently, researchers at MIT published on the "Expression of the Cloned ColE1 kil Gene in Normal and Kil.sup.r Escherichia coli, " (Altieri, M., Suit. J.L., Fan, M.-L.J. and Luria, S.E. [1986]J. Bacteriol. 168:648-654). This paper makes no mention of the ability of the prepared mutants to release periplasmic proteins into the medium. (Kobayashi et al., supra).